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ABSTRACT: Solving structures of native oligomeric protein complexes using traditional high-resolution NMR techniques remains challenging. However, increased utilization of computational platforms, and integration of information from less traditional NMR techniques with data from other complementary biophysical methods, promises to extend the boundary of NMR-applicable targets. This article reviews several of the techniques capable of providing less traditional and complementary structural information. In particular, the use of orientational constraints coming from residual dipolar couplings and residual chemical shift anisotropy offsets are shown to simplify the construction of models for oligomeric complexes, especially in cases of weak homo-dimers. Combining this orientational information with interaction site information supplied by computation, chemical shift perturbation, paramagnetic surface perturbation, cross-saturation and mass spectrometry allows high resolution models of the complexes to be constructed with relative ease. Non-NMR techniques, such as mass spectrometry, EPR and small angle X-ray scattering, are also expected to play increasingly important roles by offering alternative methods of probing the overall shape of the complex. Computational platforms capable of integrating information from multiple sources in the modeling process are also discussed in the article. And finally a new, detailed example on the determination of a chemokine tetramer structure will be used to illustrate how a non-traditional approach to oligomeric structure determination works in practice.
Journal of Structural Biology 11/2010; 173(3):515-29. · 3.41 Impact Factor
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ABSTRACT: The surfaces of mammalian cells are coated with complex carbohydrates, many terminated with a negatively charged N-acetylneuraminic acid residue. This motif is specifically targeted by pathogens, including influenza viruses and many pathogenic bacteria, to gain entry into the cell. A necessary step in the influenza virus life cycle is the release of viral particles from the cell surface; this is achieved by cleaving N-acetylneuraminic acid from cell surface glycans with a virally produced neuraminidase. We present a laboratory exercise to model this process using a glycoprotein as a glycan carrier and using real-time nuclear magnetic resonance spectroscopy to monitor N-acetylneuraminic acid release as catalyzed by neuraminidase. A time-resolved two-dimensional data-processing technique, statistical total correlation spectroscopy, enhances the resolution of the complicated one-dimensional glycoprotein spectrum and isolates characteristic peaks corresponding to substrates and products. This exercise is relatively straightforward and leads students through a wide range of biologically and chemically relevant procedures, including use of NMR spectroscopy, enzymology, and data-processing techniques.Keywords (Audience): Upper-Division Undergraduate
10/2010;
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ABSTRACT: The use of nondestructive NMR spectroscopy for enzymatic studies offers unique opportunities to identify nearly all enzymatic byproducts and detect unstable short-lived products or intermediates at the molecular level; however, numerous challenges must be overcome before it can become a widely used tool. The biosynthesis of acetyl-coenzyme A (acetyl-CoA) by acetyl-CoA synthetase is used here as a case study for the development of an analytical NMR-based time-course assay platform. We describe an algorithm to deconvolve superimposed spectra into spectra for individual molecules, and further develop a model to simulate the acetyl-CoA synthetase enzyme reaction network using the data derived from time-course NMR. Simulation shows indirectly that synthesis of acetyl-CoA is mediated via an enzyme-bound intermediate (possibly acetyl-AMP) and is accompanied by a nonproductive loss from an intermediate. The ability to predict enzyme function based on partial knowledge of the enzymatic pathway topology is also discussed.
Biophysical Journal 10/2010; 99(7):2318-26. · 3.65 Impact Factor
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ABSTRACT: The traditional NMR-based method for determining oligomeric protein structure usually involves distinguishing and assigning intra- and intersubunit NOEs. This task becomes challenging when determining symmetric homo-dimer structures because NOE cross-peaks from a given pair of protons occur at the same position whether intra- or intersubunit in origin. While there are isotope-filtering strategies for distinguishing intra from intermolecular NOE interactions in these cases, they are laborious and often prove ineffectual in cases of weak dimers, where observation of intermolecular NOEs is rare. Here, we present an efficient procedure for weak dimer structure determination based on residual dipolar couplings (RDCs), chemical shift changes upon dilution, and paramagnetic surface perturbations. This procedure is applied to the Northeast Structural Genomics Consortium protein target, SeR13, a negatively charged Staphylococcus epidermidis dimeric protein (K(d) 3.4 ± 1.4 mM) composed of 86 amino acids. A structure determination for the monomeric form using traditional NMR methods is presented, followed by a dimer structure determination using docking under orientation constraints from RDCs data, and scoring under residue pair potentials and shape-based predictions of RDCs. Validation using paramagnetic surface perturbation and chemical shift perturbation data acquired on sample dilution is also presented. The general utility of the dimer structure determination procedure and the possible relevance of SeR13 dimer formation are discussed.
Protein Science 09/2010; 19(9):1673-85. · 2.80 Impact Factor
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ABSTRACT: Residual dipolar coupling (RDC) and residual chemical shift anisotropy (RCSA) report on orientational properties of a dipolar bond vector and a chemical shift anisotropy principal axis system, respectively. They can be highly complementary in the analysis of backbone structure and dynamics in proteins as RCSAs generally include a report on vectors out of a peptide plane while RDCs usually report on in-plane vectors. Both RDC and RCSA average to zero in isotropic solutions and require partial orientation in a magnetic field to become observable. While the alignment and measurement of RDC has become routine, that of RCSA is less common. This is partly due to difficulties in providing a suitable isotopic reference spectrum for the measurement of the small chemical shift offsets coming from RCSA. Here we introduce a device (modified NMR tube) specifically designed for accurate measurement of reference and aligned spectra for RCSA measurements, but with a capacity for RDC measurements as well. Applications to both soluble and membrane anchored proteins are illustrated.
Journal of Biomolecular NMR 08/2010; 47(4):249-58. · 3.61 Impact Factor
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ABSTRACT: ADP ribosylation factors (Arfs) are N-myristoylated GTP/GDP switch proteins that have key regulatory roles in vesicle transport in eukaryotic cells. ARFs execute their roles by anchoring to membrane surfaces, where they interact with other proteins to initiate budding and maturation of transport vesicles. However, existing structures of Arf*GTP are limited to nonmyristoylated and truncated forms with impaired membrane binding. We report a high-resolution NMR structure for full-length myristoylated yeast (Saccharomyces cerevisiae) Arf1 in complex with a membrane mimic. The two-domain structure, in which the myristoylated N-terminal helix is separated from the C-terminal domain by a flexible linker, suggests a level of adaptability in binding modes for the myriad of proteins with which Arf interacts and allows predictions of specific lipid binding sites on some of these proteins.
Nature Structural & Molecular Biology 07/2010; 17(7):876-81. · 12.71 Impact Factor
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ABSTRACT: Characterization of glycosaminoglycans (GAGs), including chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS), is important in developing an understanding of cellular function and in assuring quality of preparations destined for biomedical applications. While use of (1)H and (13)C NMR spectroscopy has become common in characterization of these materials, spectra are complex and difficult to interpret when a more heterogeneous GAG type or a mixture of several types is present. Herein a method based on (1)H-(15)N two-dimensional NMR experiments is described. The (15)N- and (1)H-chemical shifts of amide signals from (15)N-containing acetylgalactosamines in CSs are shown to be quite sensitive to the sites of sulfation (4-, 6-, or 4,6-) and easily distinguishable from those of DS. The amide signals from residual (15)N-containing acetylglucosamines in HS are shown to be diagnostic of the presence of these GAG components as well. Most data were collected at natural abundance of (15)N despite its low percentage. However enrichment of the (15)N-content in GAGs using metabolic incorporation from (15)N-glutamine added to cell culture media is also demonstrated and used to distinguish metabolic states in different cell types.
Analytical Chemistry 05/2010; 82(10):4078-88. · 5.86 Impact Factor
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ABSTRACT: Long range interactions between nuclear spins and paramagnetic ions can serve as a sensitive monitor of internal motion of various parts of proteins, including functional loops and separate domains. In the case of interdomain motion, the interactions between the ion and NMR-observable nuclei are modulated in direction and magnitude mainly by a combination of overall and interdomain motions. The effects on observable parameters such as paramagnetic relaxation enhancement (PRE) and pseudocontact shift (PCS) can, in principle, be used to characterize motion. These parameters are frequently used for the purpose of structural refinements. However, their use to probe actual domain motions is less common and is lacking a proper theoretical treatment from a motional perspective. In this work, a suitable spin Hamiltonian is incorporated in a two body diffusion model to produce the time correlation function for the nuclear spin-paramagnetic ion interactions. Simulated observables for nuclei in different positions with respect to the paramagnetic ion are produced. Based on these simulations, it demonstrated that both the PRE and the PCS can be very sensitive probes of domain motion. Results for different nuclei within the protein sense different aspects of the motions. Some are more sensitive to the amplitude of the internal motion, others are more sensitive to overall diffusion rates, allowing separation of these contributions. Experimentally, the interaction strength can also be tuned by substitution of different paramagnetic ions or by varying magnetic field strength (in the case of lanthanides) to allow the use of more detailed diffusion models without reducing the reliability of data fitting.
The Journal of chemical physics 03/2010; 132(11):115102. · 3.09 Impact Factor
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ABSTRACT: Challenges for structural characterization of membrane-bound glycosphingolipids include their high internal dynamic motions and their physical proximity to membrane surfaces. Here we demonstrate that NMR paramagnetic relaxation enhancement can be used, alongside independent molecular dynamics simulations and an outer-sphere relaxation model, to quantitatively characterize the presentation (insertion depth and orientation relative to a membrane surface) of ganglioside GM1 in biologically relevant membrane environments. Longitudinal and transverse paramagnetic relaxation enhancement effects were measured for GM1, anchored to phospholipid bicelles, using both water-soluble and membrane-anchored paramagnetic probes, respectively. A method was developed to rapidly calculate paramagnetic relaxation enhancement effects from thousands of structures taken from a simulation of GM1 in a phospholipid bilayer. The combined computational and experimental approach yielded experimentally verified atomic-resolution 3D models of a highly plastic membrane-bound biomolecule.
Journal of the American Chemical Society 02/2010; 132(4):1334-8. · 9.91 Impact Factor
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ABSTRACT: The alpha-2,6-sialyltransferase (ST6Gal-I) is a key enzyme that regulates the distribution of sialic acid-containing molecules on mammalian cell surfaces. However, the fact that its native form is membrane-bound and glycosylated has made structural characterization by X-ray crystallography of this eukaryotic protein difficult. Its large size ( approximately 40 kDa for just the catalytic domain) also poses a challenge for complete structure determination by nuclear magnetic resonance (NMR). However, even without complete structure determination, there are NMR strategies that can return targeted information about select regions of the protein, including information about the active site as seen from the perspective of its bound ligands. Here, in a continuation of a previous study, a spin-labeled mimic of a glycan acceptor ligand is used to identify additional amino acids located in the protein active site. In addition, the spin-labeled donor is used to characterize the relative placement of the two bound ligands. The ligand conformation and protein-ligand contact surfaces are studied by transferred nuclear Overhauser effects (trNOEs) and saturation transfer difference (STD) experiments. The data afforded by the methods mentioned above lead to a geometric model of the bound substrates that in many ways carries an imprint of the ST6Gal-I binding site.
Biochemistry 11/2009; 48(47):11211-9. · 3.42 Impact Factor
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ABSTRACT: Sialylated forms of the Fc fragment of immunoglobulin G, produced by the human alpha2-6 sialyltransferase ST6Gal-I, were identified as potent anti-inflammatory mediators in a mouse model of rheumatoid arthritis and are potentially the active components in intravenous IgG anti-inflammatory therapies. The activities and specificities of hST6Gal-I are, however, poorly characterized. Here MS and NMR methodology demonstrates glycan modification occurs in a branch-specific manner with the alpha1-3Man branch of the complex, biantennary Fc glycan preferentially sialylated. Interestingly, this substrate preference is preserved when using a released glycan, suggesting that the apparent occlusion of glycan termini in Fc crystal structures does not dominate specificity.
Biochemistry 09/2009; 48(41):9705-7. · 3.42 Impact Factor
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ABSTRACT: Protein NMR assignments of large proteins using traditional triple resonance techniques depends on double or triple labeling of samples with (15)N, (13)C, and (2)H. This is not always practical with proteins that require expression in nonbacterial hosts. Labeling with isotopically labeled versions of single amino acids (sparse labeling) often is possible; however, resonance assignment then requires a new strategy. Here a procedure for the assignment of cross-peaks in (15)N-(1)H correlation spectra of sparsely labeled proteins is presented. It relies on the correlation of proton-deuterium amide exchange rates in native and denatured spectra of the intact protein, followed by correlation of chemical shifts in the spectra of the denatured protein with chemical shifts of sequenced peptides derived from the protein. The procedure is successfully demonstrated on a sample of a protein, Galectin-3, selectively labeled with (15)N at all alanine residues.
Journal of the American Chemical Society 05/2009; 131(14):5344-9. · 9.91 Impact Factor
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ABSTRACT: This Perspective, arising from a workshop held in July 2008 in Buffalo NY, provides an overview of the role NMR has played
in the United States Protein Structure Initiative (PSI), and a vision of how NMR will contribute to the forthcoming PSI-Biology
program. NMR has contributed in key ways to structure production by the PSI, and new methods have been developed which are
impacting the broader protein NMR community.
Journal of Structural and Functional Genomics 03/2009; 10(2):101-106.
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ABSTRACT: ADP-ribosylation factors (ARFs) are small (21 kDa), monomeric GTPases that are important regulators of membrane traffic. When membrane bound, they recruit soluble adaptors to membranes and trigger the assembly of coating complexes involved in cargo selection and vesicular budding. N-myristoylation is a conserved feature of all ARF proteins that is required for its biological functions, although the mechanism(s) by which the myristate acts in ARF functions is not fully understood. Here we present the structure of a myristoylated ARF1 protein, determined by solution NMR methods, and an assessment of the influence of myristoylation on association of ARF1.GDP and ARF1.GTP with lipid bilayers. A model in which myristoylation contributes to both the regulation of guanine nucleotide exchange and stable membrane association is supported.
Structure 02/2009; 17(1):79-87. · 6.35 Impact Factor
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ABSTRACT: Glycans that are either N-linked to asparagine or O-linked to serine or threonine are the hallmark of glycoproteins, a class of protein that dominates the mammalian proteome. These glycans perform important functions in cells and in some cases are required for protein activity. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for studying glycan structure and interactions, particularly in a form that exploits heteronuclei such as 13C. Here an approach is presented that that uses alpha-2,6-sialyltransferase (ST6Gal-I) to enzymatically add 13C-N-acetylneuraminic acid (NeuAc or sialic acid) to glycoproteins after their preparation using nonbacterial hosts. ST6Gal-I is itself a glycoprotein, and in this initial application, labeling of its own glycans and observation of these glycans by NMR are illustrated. The catalytic domain from rat ST6Gal-I was expressed in mammalian HEK293 cells. The glycans from the two glycosylation sites were analyzed with mass spectrometry and found to contain sialylated biantennary structures. The isotopic labeling approach involved removal of the native NeuAc residues from ST6Gal-I with neuraminidase, separation of the neuramindase with a lectin affinity column, and addition of synthesized 13C-CMP-NeuAc to the desialylated ST6Gal-I. Chemical shift dispersion due to the various 13C-NeuAc adducts on ST6Gal-I was observed in a 3D experiment correlating 1H-13C3-13C2 atoms of the sugar ring.
Journal of the American Chemical Society 10/2008; 130(36):11864-5. · 9.91 Impact Factor
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ABSTRACT: The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an alpha-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.
Biochemistry 10/2008; 47(36):9428-46. · 3.42 Impact Factor
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ABSTRACT: The determination of the location and conformation of a natural ligand bound to a protein receptor is often a first step in the rational design of molecules that can modulate receptor function. NMR observables, including NOEs, often provide the basis for these determinations. However, when ligands are carbohydrates, interactions mediated by extensive hydrogen-bonding networks often reduce or eliminate NOEs between ligand and protein protons. In these cases, it is useful to look to other distance- and orientation-dependent observables that can constrain the geometry of ligand-protein complexes. Here we illustrate the use of paramagnetism-based NMR constraints, including pseudo-contact shifts (PCS) and field-induced residual dipolar couplings (RDCs). When a paramagnetic center can be attached to the protein, field-induced RDCs and PCS reflect only bound-state properties of the ligand, even when averages over small fractions of bound states and large fractions of free states are observed. The effects can also be observed over a long range, making it possible to attach a paramagnetic center to a remote part of the protein. The system studied here is a Galectin-3-lactose complex. A lanthanide-binding peptide showing minimal flexibility with respect to the protein was integrated into the C terminus of an expression construct for the Galectin-3-carbohydrate-binding domain. Dysprosium ion, which has a large magnetic susceptibility anisotropy, was complexed to the peptide, making it possible to observe both PCSs and field-induced RDCs for the protein and the ligand. The structure determined from these constraints shows agreement with a crystal structure of a Galectin-3-N-acetyllactosamine complex.
Protein Science 08/2008; 17(7):1220-31. · 2.80 Impact Factor
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ABSTRACT: Relaxation rates in NMR are usually measured by intensity modulation as a function of a relaxation delay during which the relaxation mechanism of interest is effective. Other mechanisms are often suppressed during the relaxation delay by pulse sequences which eliminate their effects, or cancel their effects when two data sets with appropriate combinations of relaxation rate effects are added. Cross-correlated relaxation (CCR) involving dipole-dipole and CSA interactions differ from auto-correlated relaxation (ACR) in that the signs of contributions can be changed by inverting the state of one spin involved in the dipole-dipole interaction. This property has been exploited previously using CPMG sequences to refocus CCR while ACR evolves. Here we report a new pulse scheme that instead eliminates intensity modulation by ACR and thus allows direct measurement of CCR. The sequence uses a constant time relaxation period for which the contribution of ACR does not change. An inversion pulse is applied at various points in the sequence to effect a decay that depends on CCR only. A 2-D experiment is also described in which chemical shift evolution in the indirect dimension can share the same constant period. This improves sensitivity by avoiding the addition of a separate indirect dimension acquisition time. We illustrate the measurement of residue specific CCR rates on the non-myristoylated yeast ARF1 protein and compare the results to those obtained following the conventional method of measuring the decay rates of the slow and fast-relaxing (15)N doublets. The performances of the two methods are also quantitatively evaluated by simulation. The analysis shows that the shared constant-time CCR (SCT-CCR) method significantly improves sensitivity.
Journal of Magnetic Resonance 08/2008; 193(1):23-31. · 2.14 Impact Factor
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ABSTRACT: The solution structure of Alg13, the glycosyl donor-binding domain of an important bipartite glycosyltransferase in the yeast Saccharomyces cerevisiae, is presented. This glycosyltransferase is unusual in that it is active only in the presence of a binding partner, Alg14. Alg13 is found to adopt a unique topology among glycosyltransferases. Rather than the conventional Rossmann fold found in all GT-B enzymes, the N-terminal half of the protein is a Rossmann-like fold with a mixed parallel and antiparallel beta sheet. The Rossmann fold of the C-terminal half of Alg13 is conserved. However, although conventional GT-B enzymes usually possess three helices at the C terminus, only two helices are present in Alg13. Titration of Alg13 with both UDP-GlcNAc, the native glycosyl donor, and a paramagnetic mimic, UDP-TEMPO, shows that the interaction of Alg13 with the sugar donor is primarily through the residues in the C-terminal half of the protein.
Structure 07/2008; 16(6):965-75. · 6.35 Impact Factor
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ABSTRACT: Protein oligomerization serves an important function in biological processes, yet solving structures of protein oligomers has always been a challenge. For solution NMR, the challenge arises both from the increased size of these systems and, in the case of homo-oligomers, from ambiguities in assignment of intra- as opposed to intersubunit NOEs. In this study, we present a residual dipolar coupling (RDC)-assisted method for constructing models of homo-oligomers with purely rotational symmetry. Utilizing the fact that one of the principal axes of the tensor describing the alignment needed for RDC measurement is always parallel to the oligomer symmetry axis, it is possible to greatly restrict possible models for the oligomer. Here, it is shown that, if the monomer structure is known, all allowed dimer models can be constructed using a grid search algorithm and evaluated based on RDC simulations and the quality of the interface between the subunits. Using the Bacillus subtilis protein YkuJ as an example, it is shown that the evaluation criteria based on just two sets of NH RDCs are very selective and can unambiguously produce a model in good agreement with an existing X-ray structure of YkuJ.
Protein Science 06/2008; 17(5):899-907. · 2.80 Impact Factor
